It is largely unknown how evolutionary divergence at the level of biological molecules gives rise to different developmental processes and phenotypes. To address important but unanswered questions in evolutionary developmental biology, the proposed research will study the evolution of development by combining molecular, evolutionary, and biochemical analyses of biological molecules with phenotypic and functional analyses in an animal system. Because the evolution of developmental systems was largely propelled by the evolution of the underlying transcriptional machineries, this research will focus on the evolution of transcriptional regulation by investigating the molecular and functional evolution of a model transcription factor, bicoid. bicoid is a major anterior-posterior body axis determinant in Drosophila melanogaster. This essential gene acts as the master regulator of the segmentation gene regulatory network. bicoid was created by a lineage-specific tandem gene duplication of a derived Hox3 gene, zerknullt, and its sequence and functional conservation is restricted to the cyclorrhaphan lineage of flies. It is not known how Bicoid evolved its novel function and became an essential gene in AP axis patterning, but the acquisition of its novel DNA binding specificity may play a key role. This proposed research first sets out to investigate the molecular mechanisms that mediated the evolution of Bicoid's novel DNA binding specificity by explicitly resurrecting ancestral homeodomain sequences at the key nodes of Bicoid evolution and functionally testing their DNA binding specificities. Then, this proposed research will study the genetic and biophysical mechanisms that led to Bicoid's novel DNA-binding function. This will provide a detailed genetic and biophysical explanation of the mode and mechanisms of a major molecular innovation. To address how molecular evolutionary processes impact the evolutionary trajectory of the fly pattern determination system, this proposed research will integrate ancestral homeodomain sequences and the key mutational variants into the Drosophila melanogaster genome. Then, this proposed research will characterize the effects of the transgenic ancestral sequences by measuring their developmental functions at larval stages, determining their abilities to activate candidate gene expression, and probing their genomic DNA binding profiles. This will provide an in-depth understanding of the evolution of the genetic architecture governing a pattern determination system. Together, the proposed research will uncover how a novel critical developmental factor came to exist by exploring the evolutionary mechanisms across multiple layers of a biological system.